Murine embryonal carcinoma (EC) cells, undifferentiated stem cells of teratocarcinoma, share many features with embryonic stem cells. See, for example, Strickland, S., 1981, Cell Vol. 24: 277-278. One such property is that they are refractory to infection by viruses such as retroviruses and papovaviruses as shown by Teich, N.M., Weiss, R.A., Martin, G.R. & Lowy, D.R. (1977) Cell Vol. 12: 973-982; Swartzendruber, D.E. & Lehman, J.M. (1975) J. Cell. Physiol. Vol. 85: 179-188. This regulation appears to be primarily at the level of transcription, see Gorman, C.M., Rigby, P.W.J. & Lane, D.P. (1985) Cell Vol. 42: 519-526, although posttranscriptional blocks have also been proposed by Segal, S. & Khoury, G. (1978) Proc. Natl. Acad. Sci. U.S.A. Vol. 76: 5611-5615.
Polyomavirus host-range mutants that can propagate in EC cells have been found to contain point mutations and/or rearrangements in the viral enhancer region, suggesting that the wild-type viral enhancer does not normally function in EC cells. For a review, see Amati, P. (1985) Cell Vol. 43: 561-562. Further, it has been proposed by Linney, E., Davis, B., Overhauser, J. & Fan, H. (1985) Nature (London) Vol. 308: 470-472, that the Moloney murine leukemia virus (Mo-MuLV) regulatory sequence acting as an enhancer element in differentiated cells does not function in EC cells.
A previous report by the present inventor, Taketo, M., with collaborators Gilboa, E. & Sherman, M.I. (1985) Proc. Natl. Acad. Sci. U.S.A. Vol. 82: 2422-2426, describes the isolation of clonal transductant EC cell lines that express the neomycin-resistance gene neo linked to the Mo-MuLV long terminal repeat (LTR). The integrated recombinant viral gene is stably expressed despite the fact that these EC cells remain undifferentiated. The lack of helper provirus expression and the results of superinfection studies set forth in this reference suggest that the expression of the neo gene in the EC lines is due to cis-acting mechanisms. It is a discovery of the present invention that in at least one such transductant EC cell line, the LTR-linked neo gene is expressed because the provirus was integrated in the vicinity of a cellular enhancer. This cellular enhancer is active in EC cells and causes the neo gene to be transcribed from the bona fide LTR promoter. On the other hand, the natural enhancer of the LTR has little activity in EC cells.
A recent paper by Barklis, E., Mulligan, R.C., and Jaenisch, R. (1986) Cell Vol. 47: 391-399 characterizes recombinant proviruses expressed in F9 cells. Expression of some of the proviruses was mediated by the 5' flanking cellular sequences. It is possible that these flanking sequences contain cellular enhancer elements, unrecognized, unknown or unreported by the authors, similar to the novel cellular enhancer of the present invention. The present inventor has no further knowledge of this work and believes he is the first to identify, isolate, sequence, and propagate a novel and potentially powerful cellular enhancer operative in undifferentiated embryonic stem cells for transcription and expression of exogenous recombinant genes.